N. gonorrhoeae (GC) infects the mucosal epithelium of the human urogenital tract. It adheres to and invades epithelial cells in a multistep manner. Several bacterial ligands and their cognate epithelial cell receptors have been identified, and the initial events in adhesion and invasion are beginning to be understood. GC next traverses the epithelial cell and exits into the subepithelial matrix. This is a slow process requiring 36 - 48 hours, and the molecular mechanisms underlying it are unknown. We are interested in GC transcellular trafficking, or transcytosis, and have taken a genetic approach to study the process. We first adapted the polarized T84 epithelial cell system as a model epithelial barrier to study GC transcytosis. We then used this system to screen a random bank of mTn-generated GC mutants for fast-trafficking mutants. Four mutants with mTn insertions in three genetic loci were identified in this initial screen. Backcrosses of these mutations show that the fast- trafficking phenotype segregated with the mTn insertion. These mutants do not adhere to or invade cells more quickly, nor do they affect the integrity of the epithelial barrier. These mutants are therefore aberrant in the transcellular trafficking process, not in the initial steps of colonization. Preliminary studies indicate that the loci are likely to play a regulatory role in transcytosis. One mutant is deregulated in its growth within two types of human epithelial cells; its extracellular growth in liquid medium is normal. In this grant application, we propose to further characterize these three loci in order to elucidate their role in GC transcellular trafficking. We also propose to screen the rest of the GC mutant bank for additional fast-trafficking mutants and to characterize their mutated genes. Such studies should shed light on the genetic regulation of the transcytosis process, and hopefully guide the design of novel pharmacologic agents against intracellular GC.